Several types of secretions are produced in the secretory units. In a recent paper (Odeblad 1994a) it was described how the various contributions to the bulk mucus were identified. We denote these types G, L, S, P, F and Z, but there are also subtypes within each of them. They all originate from different preference areas with the respective secretory units or crypts. The functions of the various mucus types are described in Odeblad (1996).

The subtypes of P mucus are particularly important and Figure 3 gives information on this. In regularly spread-out samples the P mucus types P6 (with 6-fold symmetry) and P2 (with 2-fold mirror symmetry) dominate. Mucus P2 tends to adhere to mucolytic grains (Z granules) and is therefore also called P_a (mucolytically active). In very thin spread-out samples the P_p varieties ('p' for polymorphism) dominate, especially P_t with triangular crystalline formations, P_y with Y-like branching, Ph with honeycomb-like patterns, Pu ( 'u' for unbranched) and Pi with several irregular patterns.

Subtypes of P Miucus

Regular spread-out samples	Very thin samples

Fig. 3. Subtypes of P mucus are divided into two groups, those in regularly spread--out specimens and those in very thin smears. The former are best examined at x 100 - 200 magnification, the latter group at x 1000 magnification.

There are, in principle, three simple methods available for obtaining samples to study the functional anatomy and functional state of the cervix in the living woman:

(1) aspiration of the contents of single crypts;

(2) the "spread-out" method, i.e. extracting bulk mucus and then separating mechanically its constituents on a slide;

(3) the "cervical print" which enables "cervical mapping", a method which will receive special attention in this paper because it has not been described earlier.

A comparison between the three methods will be made and also how the principles may be combined to give useful information. The three methods and their combinations are all at the level of simplicity for a general medical practitioner or a practising gynaecologist because the samples obtained can be successfully examined in a microscope. However, more complicated and sophisticated investigations can also be undertaken such as electron microscopy, element analysis, electron spin resonance (ESR) and nuclear magnetic resonance (NMR) studies for research purposes.

Fig. 4. This figure illustrates how a single-crypt specimen is taken and how it can be investigated.

Early attempts to aspirate mucus from single crypts were made already in 1958, a first publication occurring some years later (Odeblad 1964). In this last study, single-crypt specimens were investigated with NMR on the microscale. This method is non-destructive and samples could then be examined and typed under the microscope. The NMR studies turned out to be important because they made it possible to identify mucus types with different viscosity, now called G, L and S (Höglund and Odeblad 1977). Figure 4 gives information on sampling and investigations. Figures 5-9 show how the different types appear under the microscope. Recent results with electron microscopy on single-crypt specimens (Menarguez 1998) are exemplified in Figure 10.

Figs. 5-7. Showing cell and crystal patterns of mucus types G (Fig. 5, x 570), L (Fig. 6, x 110), and S (Fig.7 x 430), respectively. A, sample taken from a single crypt; B, spread-out samples; C, specimens removed with the mini-swab technique. The clearest pictures are those of spread-out specimens.

Figs 8 and 9. Showing cell and crystal patterns of mucus types P6 (Fig. 8, x 110) and P2 (Fig. 9, x 110). A, sample taken from a single crypt; B, spread-out samples; C, specimens removed with the mini-swab technique. The clearest pictures are those of spread-out specimens.

Fig. 10. Scanning-electron microscopic pictures from a single-crypt sample of P mucus on a slide. In the x 130 picture (upper) the acute-angle branching of crystallite assemblies is evident. The x 1600 picture (lower) is a part of the first picture, clearly visualizing the crystallites (mainly of NaCl).

As mentioned in the Introduction, a secretary unit does not always conform with a crypt. This is illustrated in Table 1 which shows that about 30% of all single-crypt specimens are "contaminated" with undesired mucus. This may be due not only to a mixed function of a single crypt but also to simultaneous aspiration of some material from a neighbouring crypt. However, even the most precise single-crypt aspiration may sometimes contain two kinds of mucus. Table 1 also indicates that a contamination between L and S mucus is the most commonly occurring mixing. This may be due to two reasons. First, L and S mucus always occur as "neighbours", also in spread-out specimens, so their respective crypts, or "grapes" are neighbours. Second, there is a normal age process in which S crypts are successively transformed into L crypts. Samples harvested just when this transformation is occurring must therefore necessarily contain both mucus types.

The lowest parts of the cervix, especially the portio, are most accessible for single crypt sampling. Single-crypt aspiration requires good training and high skill, especially if the doctor wants to sample material from the middle or high parts of the cervix, in which case a good colposcope is a necessary instrument. Often a minute bleed may occur, making the microscopy examination difficult or even impossible. Usually the crypts in the lower part of the cervix are the G crypts thus making single crypt aspiration most important for studying the G secretion, during the infertile phases of the cycle.

The Spread-out Method

This is a most useful method to study cervical mucus. Bulk mucus is best obtained with a glass tube or a plastic tube with suction to withdraw material from the cervix, lower, middle or upper part, whatever is indicated. The sample is placed on a slide and quickly spread out in all directions (Fig. 11) with a needle and allowed to dry. Sometimes it is useful to divide the sample on two or more slides. The different crystal and cell patterns are then identified in the microscope (Figs. 5-9). The patterns for G, L and S mucus were first published as drawings (Odeblad et al 1983). At that time we believed that the present-day P mucus was a special form of S mucus. The technique, pitfalls and advantages of the spread-out technique have recently been reported (Odeblad 1994b). Mucus from secretory active "grapes" is also present. It is believed that "grape" mucus may give rise to areas in the spread-out mucus sample, not possible to classify as S, L, G, P or F.

Figure 11. This drawing indicates how a bulk cervical mucus sample is extracted and spread out on a slide for microscopic inspection.

The spread-out technique is most suited for measuring the proportions of the various mucus types. Normally there is a typical cyclic variation of all the mucus types (see, for example, Odeblad 1994b). In cases of a scanty mucus symptom such measurements can be undertaken to diagnose the presence of, for example, hyposecretion of L or S mucus and suitable therapy may be instituted.

The spread-out specimens can also be the subject of a variety of scientific investigations. Table 2 illustrates such possibilities. Investigations with X-ray spectra of elementary composition were made (Temprano et al 1994), and in other material the presence of various elements was determined with NMR (Odeblad, unpublished data, 1996). The results of these two studies are brought together in Table 2. The results of both investigations tend to agree, but the proportions between Na and Cl are systematically different. This difference may be due to difficulties in making accurate measurements. NMR measurement of Cl is especially very difficult because of the very weak NMR signals.

Phosphorus is not included in Table 2. In some samples of G and P mucus faint NMR signals of phosphorus could be observed, corresponding to 0.1 - 0.4 per cent of total mineral contents. Also, especially in Pmucus, the phosphorus signals may show a slitting indicating the presence of phosphorus as P_i, ADP, ATP, P-DNA or P-RNA. Further studiees are needed to clarify this question.

Much qualitative information can be obtained from spread-out specimens, e.g. the length and ramification of the S mucus filaments, the presence of mucolysis, and in post-coital samples also how sperm have propagated in cervical mucus. P mucus is present as many subtypes. We still do not know the functions of the various subtypes, and this is an interesting area for future research.